Vaccine to control equine protozoal myeloencephalitis in horses

ABSTRACT

The present invention provides vaccines and methods for making the vaccines that actively or passively protect an equid or other animal against  Sarcocystis neurona.  In particular, the present invention provides vaccines that provide active immunity which comprise a polypeptide or DNA vaccine that contains or expresses at least one epitope of an antigen that has an amino acid sequence substantially similar to a unique 16 (±4) kDa antigen and/or 30 (±4) kDa antigen of  Sarcocystis neurona.  The present invention further provides a vaccine that provides passive immunity to  Sarcocystis neurona  comprising polyclonal or monoclonal antibodies against at least one epitope of an antigen substantially similar to a unique 16 (±4) kDa antigen and/or 30 (±4) kDa antigen of  Sarcocystis neurona.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of copending application(s) applicationSer. No. 09/513,086 filed Feb. 24, 2000.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/152,193, filed on Sep. 2, 1999.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to vaccines and methods for making thevaccines that actively or passively protect an equid or other animalagainst Sarcocystis neurona. In particular, the present inventionrelates to vaccines that provide active immunity which comprise apolypeptide or DNA vaccine that contains or expresses at least oneepitope of an antigen that has an amino acid sequence substantiallysimilar to a unique 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona. The present invention further relates to a vaccinethat provides passive immunity to Sarcocystis neurona comprisingpolyclonal or monoclonal antibodies against at least one epitope of anantigen substantially similar to a unique 16 (±4) kDa antigen and/or 30(±4) kDa antigen of Sarcocystis neurona.

(2) Description of Related Art

Equine protozoal myeloencephalitis (EPM) is an emerging neurologicaldisease caused by the protozoan parasite Sarcocystis neurona. In recentyears, EPM has caused significant health, economic, and emotional coststo horses and their owners (reviewed by McKay et al., Veterinary Clinicsof North America 13:79-96 (1997). Opossums have been implicated as thenatural reservoir of Sarcocystis neurona because the sexual stages ofthe parasite occur in the intestines of the opossum and the sporocystsare passed in the feces of the opossum. Horses accidentally eat theopossum feces containing the sporocysts when they are grazing; however,because Sarcocystis neurona does not appear to form mature tissue cystsin equids, equids are considered to be dead end hosts. Because opossumsare ubiquitous in the United States, large numbers of equids are exposedto this parasite: approximately 50 to 60% of the horses natiowide(Blythe et al., J. Am. Vet. Med. Assoc. 210:525-527 (1997), Saville etal., J. Am. Vet. Assoc. 210:519-524 (1997), Bentz et al., J. Am. Vet.Med. Assoc. 210:517-518 (1997)).

Currently, there are no adequate diagnostic tests for determiningwhether an equid is currently infected with Sarcocystis neurona. AWestern blot test was developed to detect antibodies to Sarcocystisneurona in cerebrospinal fluid of equids suspected of having EPM;however, these Western blot assays have not been reliable in predictingthe presence of Sarcocystis neurona due to the prevalence in equids ofcross-reacting antibodies to other Sarcocystis species (Granstom et al.J. Vet. Diag. Invest. 5:88-90 (1993), Fenger et al., Vet. Parasitol.68:199-213 (1997), Bentz et al., ibid., Saville et al., ibid., Blythe etal., ibid.).

Currently, there are no vaccines to protect equids from the parasite,and current treatment regimens are effective in only about 50% of theequids (Martenuik et al., Proceedings, Conference of Research Workers onAnimal Disease, Chicago, Ill., 1997). However, these studies ontreatment efficacy were based on a low number of horses. The U.S.Department of Agriculture (USDA), Animal and Plant Health InspectionService (APHIS), National Animal Health Monitoring System (NAHMS) of theNeeds Assessment Survey (NAS) has designated EPM as one of the top twoinfectious diseases of national importance to the horse industry. Amongveterinarians and race horse owners, EPM has been ranked as the leadinghealth care concern. In particular, 58% of the race horse owners rankedEPM as the top health care concern.

Since there are no vaccines for EPM and EPM is a significant healthconcern of the equine industry, considerable effort has been directedtowards developing therapeutic methods for treating EPM. For example,U.S. Pat. No. 5,935,591 to Rossignol et al. describes using thiazolidesas a treatment for EPM; U.S. Pat. No. 5,883,095 to Granstrom et al.describes using triazine-based anti-coccidials as a treatment for EPM;U.S. Pat. No. 5,830,893 to Russel describes using triazinediones as atreatment for EPM; U.S. Pat. No. 5,747,476 to Russel describes using acombination of pyrimethamine and a sulfonamide, preferably sulfadiazinein the absence of known therapeutic amounts of trimethoprim as atreatment for EPM; and U.S. Pat. No. 5,925,622 to Rossignol et al.describes using aryl glucuronide of 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide as a treatment for EPM.

Treatment for EPM is expensive and cumbersome because of the longduration required to achieve positive results. Because many horsescannot be successfully treated, economically and emotionally valuableanimals have been lost to EPM. However, the extent of EPM's economicimpact is even greater because of the large sums of money spent by horseowners for treating lame horses which have been incorrectly diagnosedwith EPM, for giving prophylactic treatments that have no scientificbasis, and for finding positive post-race drug test results.

EPM has been the cause of hysteria in the equid industry. The smallamount of scientific data available on EPM supports a high exposure rateof equids, but there are no data available that document the rate ofclinical disease resulting from exposure to the parasite. Because ofthis, horse owners and veterinarians assume that the rate of clinicaldisease is high. As a result, several alarming consequences have arisen.Equids with lameness or other neurological diseases are beingmisdiagnosed as having EPM. People whose livelihoods depend on horsesare resorting to medicating all their horses all of the time withantimicrobials. This approach to treating EPM is very widespread in theracing industry. However, this indiscriminate use of antimicrobials hasthe potential of leading to resistant bacteria such as Salmonella, E.coli, etc. which will then enter the environment and pose a risk forhumans and animals. Thus, the repercussions of EPM may extend beyond adisease that merely affects the horse industry. All of the repercussionsof EPM are expensive, decrease the value realized to the U.S. equidindustry, and raise the specter of a public health problem of immenseproportions.

Therefore, there is a need for a treatment of EPM that is effective andhas little or no side-effects.

SUMMARY OF THE INVENTION

The present invention provides vaccines and methods for making thevaccines that protect an equid or other animal host against Sarcocystisneurona. In particular, the present invention provides a vaccine thatelicits active immunity against Sarcocystis neurona which contains atleast one epitope of a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona. The present invention further provides a DNAvaccine that elicits active immunity against Sarcocystis neuronacomprising a DNA encoding at least one epitope of a 16 (±4) kDa antigenand/or 30 (±4) kDa antigen of Sarcocystis neurona.

The present invention further provides a vaccine for providing passiveimmunity to a Sarcocystis neurona infection comprising antibodies whichare against at least one epitope of a 16 (±4) kDa antigen and/or 30 (±4)kDa antigen of Sarcocystis neurona. In particular, a vaccine wherein theantibodies are selected from the group consisting of polyclonalantibodies and monoclonal antibodies against a 16 (±4) kDa antigenand/or 30 (±4) kDa antigen of Sarcocystis neurona. In a preferredembodiment of the vaccine, the vaccine is provided in a pharmaceuticallyaccepted carrier.

Further, the present invention further provides a vaccine for activeimmunization of an equid against a Sarcocystis neurona infectioncomprising an antigen containing at least one epitope of a 16 (±4) kDaantigen and/or 30 (±4) kDa antigen of Sarcocystis neurona. In oneembodiment of the present invention, the antigen is a recombinantpolypeptide produced in a plasmid in a microorganism other thanSarcocystis neurona, preferably, in an E. coli. In a preferredembodiment, the vaccine is provided in a pharmaceutically acceptedcarrier.

Further, the present invention provides for a vaccine wherein the 16(±4) kDa antigen and/or 30 (±4) kDa antigen of Sarcocystis neuronaantigen is provided as a fusion polypeptide wherein an amino end and/ora carboxyl end of the antigen is fused to all or a portion of apolypeptide that facilitates the isolation of the antigen from themicroorganism in which the antigen is produced. In a preferredembodiment, the polypeptide is selected from the group consisting ofglutathione S-transferase, protein A, maltose binding protein, andpolyhistidine.

The present invention also provides a vaccine for protecting an equidfrom a Sarcocystis neurona infection comprising a DNA that encodes atleast one epitope of a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona. In a preferred embodiment, the DNA is operablylinked to a promoter to enable transcription of the DNA in the cell ofan equid. Preferably, the vaccine is provided in a pharmaceuticallyaccepted carrier.

The present invention further provides a method for vaccinating an equidagainst a Sarcocystis neurona infection comprising: (a) providing arecombinant antigen of the Sarcocystis neurona produced from amicroorganism culture wherein the microorganism contains a DNA thatencodes a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen of Sarcocystisneurona; and (b) vaccinating the equid. Preferably, the vaccine is in apharmaceutically accepted carrier.

In a preferred embodiment of the method, the recombinant antigen is afusion polypeptide which is fused at the amino terminus and/or carboxylterminus to a polypeptide that facilitates the isolation of therecombinant antigen. In particular, the polypeptide is all or a portionof the polypeptide selected from the group consisting of glutathioneS-transferase, protein A, maltose binding protein, and polyhistidine.Further, the method includes producing the antigen from a DNA which isin a plasmid in a microorganism wherein the DNA is operably linked to apromoter which enables transcription of the DNA to produce therecombinant antigen for the vaccine.

The present invention further provides a method for vaccinating an equidagainst a Sarcocystis neurona infection comprising: (a) providing in acarrier solution a DNA in a plasmid which encodes at least one epitopeof a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen of Sarcocystisneurona; and (b) vaccinating the equid with the DNA in the carriersolution. Preferably, the DNA is in a carrier solution that ispharmaceutically accepted for DNA vaccines. In a preferred embodiment,the DNA is operably linked to a promoter to enable transcription of theDNA in a cell of the equid.

The present invention further provides a method for providing passiveimmunity to a Sarcocystis neurona infection in an equid comprising: (a)providing antibodies selected from the group consisting of polyclonalantibodies and monoclonal antibodies which are against at least oneepitope of a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona; and (b) inoculating the equid. Preferably, theantibodies are provided in a pharmaceutically accepted carrier.

Further still, the present invention provides a method for producing anantigen comprising: (a) providing a microorganism in a culturecontaining a DNA encoding a fusion polypeptide comprising at least oneepitope of a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona and a polypeptide that facilitates isolation of thefusion polypeptide; (b) culturing the microorganism in a culture toproduce the fusion polypeptide; and (c) isolating the fusionpolypeptide. In one embodiment, the fusion polypeptide is isolated byaffinity chromatography which can be affinity chromatography thatcomprises an IgG-linked resin when the polypeptide consists of all or aportion of protein A, an Ni²⁺ resin when the polypeptide ispolyhistidine, amylose resin when the polypeptide is all or part of themaltose binding protein, or glutathione Sepharose 4B resin when thepolypeptide is all or part of glutathione S-transferase.

Further still, the present invention provides a method for producing anantibody comprising: (a) providing a microorganism in a culturecontaining a DNA encoding a fusion polypeptide comprising at least oneepitope of a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona linked to a polypeptide that facilitates isolationof the fusion polypeptide; (b) culturing the microorganism in a cultureto produce the fusion polypeptide; (c) isolating the fusion polypeptide;(d) producing the antibody from the polypeptide. In a preferredembodiment, the polypeptide is removed from the antigen portion of thefusion polypeptide.

And further still, the present invention provides a method for producinga monoclonal antibody comprising: (a) providing a microorganism in aculture containing a DNA encoding a fusion polypeptide comprising atleast one epitope of a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona linked to a polypeptide that facilitates isolationof the fusion polypeptide; (b) culturing the microorganism in a cultureto produce the fusion polypeptide; (c) isolating the fusion polypeptide;and (d) producing the monoclonal antibody from the polypeptide.Preferably, the polypeptide is removed from the antigen portion of thefusion polypeptide.

The present invention comprises a vaccine for an equid comprising anisolated recombinant protein encoded by a cDNA produced from RNA ofSarcocystis neurona encoding a 16 (±4) kDa antigen and/or 30 (±4) kDaantigen, and a vaccine carrier. In another embodiment of the presentinvention, the vaccine for an equid comprises a recombinant virus vectorcontaining DNA encoding a 16 (±4) kDa antigen and/or 30 (±4) kDa antigenof Sarcocystis neurona, and a vaccine carrier. In particular, therecombinant virus is selected from the group consisting of equineherpesvirus, vaccinia virus, canary pox virus, raccoon poxvirus,adenovirus, and baculovirus. In an embodiment further still, the presentinvention comprises a DNA vaccine for an equid comprising a plasmidcontaining DNA encoding a 16 (±4) kDa antigen and/or 30 (±4) kDa antigenof Sarcocystis neurona.

The present invention provides a method for protecting an equid againstSarcocystis neurona which comprises providing a vaccine that wheninjected into the equid causes the equid to produce antibodies and cellmediated immunity against a 16 (±4) kDa antigen and/or 30 (±4) kDaantigen of the Sarcocystis neurona wherein the antibodies preventinfection by the Sarcocystis neurona. In particular, the vaccinecomprises the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen in avaccine carrier. The present invention further provides a vaccinecomprising a recombinant virus vector that expresses the 16 (±4) kDaantigen and/or 30 (±4) kDa antigen. In particular, the recombinant virusvector is selected from the group consisting of equine herpesvirus,vaccinia virus, canary pox virus, raccoon poxvirus, and adenovirus. Thepresent invention further still provides a vaccine which comprises a DNAplasmid encoding the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen.

The present invention further comprises a monoclonal antibody thatselectively binds to a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona. The present invention also comprises an isolatedrecombinant protein encoded by a cDNA produced from RNA of Sarcocystisneurona encoding a protein which is a 16 (±4) kDa antigen and/or 30 (±4)kDa antigen. Thus, the present invention further comprises an isolatedDNA that encodes a 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona. Finally, the present invention comprises abacterial clone containing a plasmid comprising a DNA encoding a 16 (±4)kDa antigen and/or 30 (±4) kDa antigen of Sarcocystis neurona. Inparticular, the bacterial clone expresses the 16 (±4) kDa antigen and/or30 (±4) kDa antigen of Sarcocystis neurona.

It is therefore an object of the present invention to provide a vaccinefor the prophylactic or therapeutic treatment of protozoalmyeloencephalitis in equids. In particular, it is an object of thepresent invention to provide a vaccine for providing active immunityagainst Sarcocystis neurona which comprises a 16 (±4) kDa antigen and/or30 (±4) kDa antigen of Sarcocystis neurona.

It is also an object of the present invention to provide a vaccine thatprovides passive immunity in an equid against Sarcocystis neurona whichcomprises antibodies against a 16 (±4) kDa antigen and/or 30 (±4) kDaantigen of Sarcocystis neurona.

These and other objects of the present invention will becomeincreasingly apparent by reference to the following embodiments.

DESCRIPTION OF PREFERRED EMBODIMENTS

The following definitions are provided herein to promote a betterunderstanding of the present invention.

The term “antibody” as used herein refers to an immunoglobulin moleculewith the capacity to bind with a specific antigen as the result of aspecific immune response. Immunoglobulins are serum proteins made up oflight and heavy polypeptide chains and divisible into classes, whichcontain within them antibody activities toward a wide range of antigens.

The term “polyclonal antibody” as used herein refers to a mixedpopulation of antibodies made against a particular pathogen or antigen.In general, the population contains a variety of antibody groups, eachgroup directed towards a particular epitope of the pathogen or antigen.To make polyclonal antibodies, the whole pathogen or an isolated antigenis introduced by inoculation or antigen. To make polyclonal antibodies,the whole pathogen or an isolated antigen is introduced by inoculationor infection into a host which induced the host to make antibodiesagainst the pathogen or antigen.

The term “monoclonal antibody” as used herein refers to antibodiesproduced by a single line of hybridoma cells all directed towards oneepitope on a particular antigen. The antigen used to make the monoclonalantibody can be provided as an isolated protein of the pathogen or thewhole pathogen. A hybridoma is a clonal cell line that consists ofhybrid cells formed by the fusion of a myeloma cell and a specificantibody-forming cell. In general, monoclonal antibodies are of mouseorigin; however, monoclonal antibody also refers to a clonal populationof an antibody made against a particular epitope of an antigen producedby phage display technology or method that is equivalent to phagedisplay or hybrid cells of non-mouse origin.

The term “antigen” as used herein refers to a substance which stimulatesproduction of antibody or sensitized cells during an immune response. Anantigen includes the whole pathogen or a particular protein of thepathogen. An antigen consists of multiple epitopes, each epitope ofwhich is capable of causing the production of an antibody against theparticular epitope.

The term “epitope” as used herein refers to an immunogenic region of anantigen which is recognized by a particular antibody molecule. Ingeneral, an antigen will possess one or more epitopes, each capable ofbinding an antibody that recognizes the particular epitope. An antibodycan recognize a contiguous epitope which is an epitope that is a linearsequence of amino acids in the antigen molecule, or a non-contiguousepitope which is an epitope that spans non-contiguous amino acids in theantigen which have been brought together because of thethree-dimensional structure of the antigen.

The term “active immunity” as used herein includes both antibodyimmunity and/or cell mediated immunity against Sarcocystis neuronainduced by vaccinating an equid with the vaccine of the presentinvention comprising the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen.

The term “passive immunity” as used herein refers to the protectionagainst Sarcocystis neurona provided to an equid as a result ofvaccinating the equid with a vaccine comprising antibodies against the16 (±4) kDa antigen and/or 30 (±4) kDa antigen.

The present invention provides a vaccine that protects equids againstSarcocystis neurona. In a preferred embodiment, the vaccine consists ofa 16 (±4) kDa antigen and/or 30 (±4) kDa antigen in a subunit vaccine.Preferably, the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen areproduced in a recombinant bacterium or eukaryote expression vector whichproduces the proteins which are then isolated to make the vaccine. Inanother embodiment of the vaccine, the vaccine is a DNA vaccine thatcomprises a recombinant DNA molecule, preferably in a plasmid, thatcomprises DNA encoding all or part of the 16 (±4) kDa antigen and/or 30(±4) kDa antigen. In another embodiment of the vaccine, the recombinantDNA is inserted into a virus vector to provide a live vaccine which is arecombinant DNA virus. In U.S. Ser. No. 09/156,954, filed on Sep. 18,1998, now U.S. Pat. No. 6,153,394 to Mansfield et al., which is herebyincorporated herein by reference, it was disclosed that Sarcocystisneurona possesses two unique antigens, a 16 (±4) antigen and a 30 (±4)kDa antigen. These antigens do not react with antibodies from otherSarcocystis spp. Thus, these antigens are useful for producing vaccinesthat protect equids against Sarcocystis neurona.

The route of administration for the vaccines of the present inventioncan include, but is not limited to, intramuscular, intraperitoneal,intradermal, subcutaneous, intravenous, intraarterial, intraocular, andoral as well as transdermal or by inhalation or suppository. Thepreferred routes of administration include intranasal, intramuscular,intraperitoneal, intradermal, and subcutaneous injection. The vaccinecan be administered by means including, but not limited to, syringes,needle-less injection devices, or microprojectile bombardment gene guns(biolistic bombardment).

The vaccines of the present invention are formulated in pharmaceuticallyaccepted carriers according to the mode of administration to be used.One skilled in the art can readily formulate a vaccine that comprisesthe polyepptide or DNA of the present invention. In cases whereintramuscular injection is preferred, an isotonic formulation ispreferred. Generally, additives for isotonicity can include sodiumchloride, dextrose, mannitol, sorbitol, and lactose. In particularcases, isotonic solutions such as phosphate buffered saline arepreferred. The formulations can further provide stabilizers such asgelatin and albumin. In some embodiments, a vasco-constriction agent isadded to the formulation. The pharmaceutical preparations according tothe present invention are provided sterile and pyrogen free. However, itis well known by those skilled in the art that the preferredformulations for the pharmaceutically accepted carrier which comprisethe vaccines of the present invention are those pharmaceutical carriersapproved in the regulation promulgated by the the United StatesDepartment of Agriculture, or equivalent government agency in a foreigncountry such as Canada or Mexico, for polypeptide, recombinant vector,antibody, and DNA vaccines intended for veterinary applications.Therefore, the pharmaceutically accepted carriers for commercialproduction of the vaccines of the present invention are those carriersthat are already approved or will at some future date be approved by theappropriate government agency in the United States of America or foreigncountry.

Inoculation of an equid is preferably by a single vaccination which inthe case of polypeptide, recombinant vector, and DNA vaccines produces afull, broad immunogenic response. In another embodiment of the presentinvention, the equid is subjected to a series of vaccinations to producea full, broad immune response. When the vaccinations are provided in aseries, the vaccinations can be provided between about 24 hours apart totwo weeks or longer between vaccinations. In certain embodiments, theequid is vaccinated at different sites simultaneously.

The vaccines of the present invention are generally intended to be aprophylactic treatment which prevents Sarcocystis neurona fromestablishing an infection in an equid. However, the vaccines are alsointended for the therapeutic treatment of equids already infected withSarcocystis neurona. For example, antibody vaccines of the presentinvention are suitable for therapeutic purposes. However, vaccines thatprovide active immunity have also been shown to be effective when givenas a therapeutic treatment against various diseases. Thus, the immunitythat is provided by the present invention can be either active immunityor passive immunity and the intended use of the vaccine can be eitherprophylactic or therapeutic.

With respect to the above, the vaccine that elicits active immunity in ahost can be a polypeptide vaccine or a DNA vaccine which produces thepolypeptide in a vaccinated host. Alternatively, the vaccine can be arecombinant microorganism vaccine that expresses the 16 (±4) kDa antigenand/or 30 (±4) kDa antigen or a recombinant virus vector that expressesthe 16 (±4) kDa antigen and/or 30 (±4) kDa antigen.

Thus, in one embodiment of the present invention, the active immunity isprovided by a vaccine that consists of the isolated 16 (±4) kDa antigenand/or 30 (±4) kDa antigen or the 16 (±4) kDa antigen and/or 30 (±4) kDaantigen as a fusion polypeptide wherein the amino and/or carboxylterminus is fused to another polypeptide, preferably a polypeptide thatfacilitates isolation of the fusion polypeptide. The fusion polypeptidecomprising the vaccine is preferably produced in vitro in an expressionsystem from a DNA that encodes the antigens which is in a microorganismsuch as bacteria, yeast, or fungi; in eukaryote cells such as amammalian or an insect cell; or, in a virus expression vector such asadenovirus, poxvirus, herpesvirus, Simliki forest virus, baculovirus,bacteriophage, or sendai virus. In particular, suitable bacterialstrains for producing the 16 (±4) kDa antigen and/or 30 (±4) kDa antigenor the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen as fusionpolypeptides includes Escherichia coli, Bacillus subtilis, or any otherbacterium that is capable of expressing heterologous polypeptides.Suitable yeast for expressing the 16 (±4) kDa antigen and/or 30 (±4) kDaantigen or 16 (±4) kDa antigen and/or 30 (±4) kDa antigen as fusionpolypeptides include Saccharomyces cerevisiae, Schizosaccharomycespombe, Candida, or any other yeast capable of expressing heterologouspolypeptides. Methods for using the aforementioned and the like toproduce recombinant polypeptides for vaccines are well known in the art.

For any of the above, transformed host cells are cultured underconditions which produce the 16 (±4) kDa antigen and/or 30 (±4) kDaantigen or the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen as fusionpolypeptides. The resulting expressed polypeptides can be isolated fromthe culture, medium or cell extracts, using purification methods such asgel filtration, affinity chromatography, ion exchange chromatography, orcentrifugation. Furthermore, the present invention further includespolypeptides that comprise only those epitopes of the 16 (±4) kDaantigen and/or 30 (±4) kDa antigen which are responsible for conferringprotective immunity against Sarcocystis neurona. It is also understoodthat antigens of other Sarcocystis spp. that correspond to the 16 (±4)kDa antigen and/or 30 (±4) kDa antigen of Sarcocystis neurona are withinthe scope of the present invention.

DNA encoding the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen can beobtained from a genome preparation of Sarcocystis neurona using apolymerase chain reaction (PCR) method that uses DNA primers whichcorrespond to the nucleotide sequences encoding the amino and carboxylends of the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen. Preferablythe 5′ ends of the primers contain a restriction enzyme site thatfacilitates the subsequent steps of constructing 16 (±4) kDa antigenand/or 30 (±4) kDa antigen expression systems. Alternatively, the DNAprimers can correspond to an internal region of the nucleotide sequenceencoding the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen forproducing a DNA encoding a particular epitope of the antigen. Primerdesign and PCR methods are well known in the art.

In a preferred embodiment, the DNA is in a plasmid and the DNA isoperably linked to a promoter which effects the expression of the 16(±4) kDa antigen and/or 30 (±4) kDa antigen in a microorganism,preferably E. coli. As used herein, the term “operably linked” meansthat the polynucleotide of the present invention and a DNA containing anexpression control sequence, e.g., transcription promoter andtermination sequences, are situated in a vector or cell such thatexpression of the antigen encoded by the polynucleotide is regulated bythe expression control sequence. Methods for cloning DNA such as the DNAencoding the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen and operablylinking DNA containing expression control sequences thereto are wellknown in the art. Expression of the 16 (±4) kDa antigen and/or 30 (±4)kDa antigen in a microorganism enables the antigen to be produced usingfermentation technologies which are used commercially for producinglarge quantities of recombinant polypeptides.

To facilitate isolation of the 16 (±4) kDa antigen and/or 30 (±4) kDaantigen produced as above, a fusion polypeptide is made wherein theantigen is linked to another polypeptide which enables isolation byaffinity chromatography. Preferably, a fusion polypeptide is made usingone of the aforementioned expression systems. Therefore, the DNAencoding the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen is linked toa DNA encoding a second polypeptide to produce a fusion polypeptidewherein the amino and/or carboxyl terminus of the antigen is fused to apolypeptide which allows for the simplified recovery of the antigen as afusion polypeptide. The fusion polypeptide can also prevent the antigenfrom being degraded during purification. While a vaccine comprising thefusion polypeptide is efficacious, in some instances it can be desirableto remove the second polypeptide after the purification. Therefore, itis also contemplated that the fusion polypeptide comprise a cleavagesite at the junction between the antigen and the polypeptide. Thecleavage site consists of an amino acid sequence that is cleaved with anenzyme specific for the amino acid sequence of that site. Examples ofsuch cleavage sites that are contemplated include the enterokinasecleavage site which is cleaved by enterokinase, the factor Xa cleavagesite which is cleaved by factor Xa, and the GENENASE cleavage site whichis cleaved by GENENASE (GENENASE is a trademark of New England Biolabs,Beverly, Mass.).

An example of a procaryote expression system for producing the 16 (±4)kDa antigen and/or 30 (±4) kDa antigen is the Glutathione S-transferase(GST) Gene Fusion System available from Amersham Pharmacia Biotech,Piscataway, N.J., which uses the pGEX-4T-1 expression vector plasmid.The DNA encoding the antigen is fused in frame with the GST gene. TheGST part of the fusion polypeptide allows the rapid purification of thefusion polypeptide using glutathione Sepharose 4B affinitychromatography. After purification, the GST portion of the fusionpolypeptide can be removed by cleavage with a site-specific proteasesuch as thrombin or factor Xa to produce a polypeptide free of the GSTgene. The antigen free of GST is produced by a second round ofglutathione Sepharose 4B affinity chromatography.

Another example for producing the 16 (±4) kDa antigen and/or 30 (±4) kDaantigen is a method which links in-frame with the gene encoding theantigen, codons that encode polyhistidine. The polyhistidine preferablycomprises six histidine residues which allows purification of the fusionpolypeptide by metal affinity chromatography, preferably nickel affinitychromatography. To produce the native antigen free of the polyhistidine,a cleavage site such as an enterokinase cleavage site is fused in framebetween the codons encoding the polyhistidine and the codons encodingthe antigen. The native polypeptide free of the polyhistidine is made byremoving the polyhistidine by cleavage with enterokinase. The antigenfree of the polyhistidine is produced by a second round of metalaffinity chromatography. This method was shown to be useful forpreparing the LcrV antigen of Y. pestis which was disclosed in Motin etal. Infect. Immun. 64:4313-4318 (1996), which is hereby incorporatedherein by reference. The Xpress System available from Invitrogen,Carlsbad, Calif. is an example of a commercial kit which is availablefor making and then isolating polyhistidine-polypeptide fusion proteins.

A method further still for producing the 16 (±4) kDa antigen and/or 30(±4) kDa antigen is disclosed by Motin et al., Infect. Immun.64:3021-3029 (1995), which is hereby incorporated herein by reference.Motin et al. disclosed a DNA encoding a fusion polypeptide consisting ofthe DNA encoding an antigen linked to DNA encoding a portion of proteinA wherein DNA encoding an enterokinase cleavage site is interposedbetween the DNA encoding protein A and the antigen. The protein Aenables the fusion polypeptide to be isolated by IgG affinitychromatography, and the antigen free of the protein A is produced bycleavage with an enterokinase. The protein A is then remove by a secondround of IgG affinity chromatography.

Another method for producing polypeptide vaccines against Sarcocystisneurona is based on methods disclosed in U.S. Pat. No. 5,725,863 toDaniels et al., which is hereby incorporated herein by reference. TheDaniels method can be used to make the 16 (±4) kDa antigen and/or 30(±4) kDa antigen vaccine of the present invention which consists of anenterotoxin which has inserted therein upwards of 100 amino acidresidues of the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen. Anothermethod that can be used to make the polypeptide vaccines of the presentinvention is disclosed in U.S. Pat. No. 5,585,100 to Mond et al., whichis hereby incorporated herein by reference, which provides methods formaking various fusion polypeptide vaccines. Further methods aredisclosed in U.S. Pat. No. 5,589,384 to Liscombe, which is herebyincorporated herein by reference. Finally, the pMAL Fusion andPurification System available from New England Biolabs is anotherexample of a method for making a fusion polypeptide wherein a maltosebinding protein is fused to the antigen. The maltose binding proteinfacilitates isolation of the fusion polypeptide by amylose affinitychromatography. The maltose binding protein can subsequently be releasedby cleavage with any of the aforementioned cleavage enzymes.

While bacterial methods are used to produce the 16 (±4 ) kDa antigenand/or 30 (±4) kDa antigen, it can be desirable to produce the antigenin a eukaryote expression system. A particularly useful system is thebaculovirus expression system which is disclosed in U.S. Pat. No.5,229,293 to Matsuura et al., which is hereby incorporated herein byreference. Baculovirus expression vectors suitable to produce theantigen are the pPbac and pMbac vectors from Stratagene; and theBac-N-Blue vector, the pBlueBac4.5 vector, pBlueBacHis2-A,B,C, and thepMelBac available from Invitrogen, Carlsbad, Calif.

Another eukaryote system useful for expressing the 16 (±4) kDa antigenand/or 30 (±4) kDa antigen is a yeast expression system such as the ESPYeast Protein Expression and Purification System available fromStratagene. Another yeast expression system is any one of thePichia-based Expression systems from Invitrogen. Mammalian expressionsystems are also embraced by the present invention. Examples ofmammalian expression systems are the LacSwitch II system, the pBKPhagemid, pXT1 vector system, and the pSG5 vector system fromStratagene; the pTargeT mammalian expression vector system, the pSImammalian expression vector, pCI mammalian expression vector, andpAdVantage vectors available from Promega Corporation, Madison, Wis.;and the Ecdysone-Inducible Mammalian Expression System, pCDM8, pcDNA1.1,and pcDNA1.1/Amp available from Invitrogen.

Another method for producing the 16 (±4) kDa antigen and/or 30 (±4) kDaantigen in a eukaryote expression system is to insert DNA encoding theantigen into the genome of a eukaryote cell or in a eukaryote virusexpression vector such as herpesvirus, poxvirus, or adenovirus to make arecombinant virus that expresses the antigen. The recombinant virusvectors are used to infect mammalian cells wherein the antigens areproduced in the cell. U.S. Pat. No. 5,223,424 to Cochran et al., whichis hereby incorporated herein by reference, provides methods forinserting genes into herpesvirus expression vectors. U.S. Pat. Nos.5,338,683 and 5,494,807 to Paoletti et al. and U.S. Pat. No. 5,935,777to Moyer et al., which are hereby incorporated herein by reference,provide methods for inserting genes into poxvirus expression vectorssuch as vaccinia virus, entomopoxvirus, and canary poxvirus. In anotherembodiment, the genes encoding the antigen can be inserted into adefective virus such as the herpesvirus amplicon vector which isdisclosed in U.S. Pat. No. 5,928,913 to Efstathiou et al., which ishereby incorporated herein by reference. In any of the aforementionedvirus vectors, the gene encoding the antigen are operably linked to aeukaryote promoter at the 5′ end of the DNA encoding the protein and aeukaryote termination signal and poly(A) signal at the 3′ end of thegene. Examples of such promoters are the cytomegalovirus immediate-early(CMV) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR)promoter, the simian virus 40 (SV40) immediate-early promoter, andinducible promoters such as the metallothionein promoter. An example ofa DNA having a termination and poly(A) signal is the SV40 late poly(A)region. Another example of a viral expression system suitable forproducing the antigen is the Sindbis Expression system available fromInvitrogen. The sue of these commercially available expression vectorsand systems are well known in the art.

While subunit vaccines comprising the 16 (±4) kDa antigen and/or 30 (±4)kDa antigen generally provide good humoral protection, it can bedesirable to provide the antigen as a component of a live recombinantvector vaccine. Therefore, the present invention further embracesrecombinant virus vector vaccines wherein DNA encoding the antigen isinserted into a recombinant virus vector. In one embodiment of therecombinant virus vector vaccine, the DNA encoding the antigen isinserted into a herpesvirus vector according to the method taught byCochran et al. in U.S. Pat. No. 5,233,424, which is hereby incorporatedherein by reference. It is particularly desirable to have a recombinantvirus vector vaccine against Sarcocystis neurona that is fetal safe,which allows the vaccine to be given to pregnant mares without affectingthe fetus. U.S. Pat. Nos. 5,741,696 and 5,731,188 to Cochran et al.,which are hereby incorporated herein by reference, teach methods formaking and using live recombinant herpesvirus vaccine vectors which arefetal safe.

Other recombinant virus vector vaccines embraced by the presentinvention, include but are not limited to, adenovirus, adeno-associatedvirus, parvovirus, and various poxvirus vectors to express the 16 (±4)kDa antigen and/or 30 (±4) kDa antigen. For example, U.S. Pat. Nos.5,338,683 and 5,494,807 to Paoletti et al. teach recombinant virusvaccines consisting of either vaccinia virus or canary poxvirusexpressing foreign antigens; and U.S. Pat. No. 5,266,313 to Esposito etal. teaches recombinant raccoon poxvirus vectors expressing foreignantigens. Therefore, the present invention embraces recombinant poxvirusvaccines that express the 16 (±4) kDa antigen and/or 30 (±4) kDa antigenmade according to the methods taught in any one of U.S. Pat. Nos.5,338,683; 5,494,807; and 5,935,777, which are hereby incorporatedherein by reference.

While the above refer to DNA sequences encoding the 16 (±4) kDa antigenand/or 30 (±4) kDa antigen, the present invention also includes RNAsequences for encoding the antigen.

The present invention further includes vaccines that comprise the 16(±4) kDa antigen and/or 30 (±4) kDa antigen or particular epitopes ofthe 16 (±4) kDa antigen and/or 30 (±4) kDa antigen as components of aheat-stable spore delivery system made according to the method taught inU.S. Pat. No. 5,800,821 to Acheson et al., which is hereby incorporatedherein by reference. Therefore, the present invention provides agenetically engineered bacterial cell containing DNA encoding the 16(±4) kDa antigen and/or 30 (±4) kDa antigen. When the recombinantbacterial spore vaccine is orally administered to the equid, the sporesgerminate in the gastrointestinal tract of the animal and the bacterialexpresses the antigen which comes into contact with the animal's immunesystem and elicits an immune response. The vaccine has the advantage ofbeing heat stable; therefore, it can be stored at room temperature foran indefinite period of time.

Another embodiment of the Sarcocystis neurona vaccine is a DNA vaccinethat elicits an active immune response in an equid. The DNA vaccineconsists of DNA having a DNA sequence substantially similar to the DNAsequence that encodes the 16 (±4) kDa antigen and/or 30 (±4) kDaantigen. The DNA encoding the antigen is operably linked at or near itsstart codon to a promoter that enables transcription of the antigen fromthe DNA when the DNA is the cells of the equid. Preferably, the DNA isin a plasmid. Promoters for expression of DNAs in DNA vaccines are wellknown in the art and include among others such promoters as the RSV LTRpromoter, the CMV immediate early promoter, and the SV40 T antigenpromoter. It is further preferred that the DNA is operably linked at theor near the termination codon of the sequence encoding antigen to a DNAfragment comprising a transcription termination signal and poly(A)recognition signal. Preferably, the vaccine is in an acceptedpharmaceutical carrier or in a lipid or liposome carrier similar tothose disclosed in U.S. Pat. No. 5,703,055 to Felgner, which is herebyincorporated herein by reference. The DNA can be provided to the equidby a variety of methods such as intramuscular injection, intrajetinjection, or biolistic bombardment. Making DNA vaccines and methods fortheir use are provided in U.S. Pat. Nos. 5,589,466 and 5,580,859, bothto Felgner, which are hereby incorporated herein by reference. Finally,a method for producing pharmaceutical grade plasmid DNA is taught inU.S. Pat. No. 5,561,064 to Marquet et al., which is hereby incorporatedherein by reference.

Therefore, using the abovementioned methods, DNA vaccines that expressthe 16 (±4) kDa antigen and/or 30 (±4) kDa antigen are made and used tovaccinate equids against Sarcocystis neurona. The advantage of the DNAvaccine is that the DNA is conveniently propagated as a plasmid which isa simple and inexpensive means for producing a vaccine, and since thevaccine is not live, the regulatory difficulties associated with gettingrecombinant virus vaccines approved are not present. One skilled in theart would appreciate that while the polypeptide produced for thepolypeptide vaccine or by the DNA vaccine can be the entire 16 (±4) kDaantigen and/or 30 (±4) kDa antigen, the present invention also includespolypeptide and DNA vaccines wherein the vaccine consists of asubfragment of the antigen which comprises one or more epitopes of theantigen or a DNA encoding one or more epitopes of the antigen.Furthermore, the polypeptide and DNA vaccines of the present inventioncan comprise synthetically produced polypeptides or DNA which are madeby chemical synthesis methods well known in the art.

While the DNA and polypeptide provided herein is from Sarcocystisneurona, the present invention further encompasses similar antigens fromother Sarcocystis spp. Thus, it is anticipated that the vaccines andmethods disclosed herein are useful for producing vaccines against otherSarcocystis spp.

In another embodiment of the present invention, the vaccine providespassive immunity to Sarcocystis neurona. A vaccine that elicits passiveimmunity against Sarcocystis neurona consists of polyclonal antibodiesor monoclonal antibodies that are against the unique 16 (±4) and/or 30(±4) antigen of Sarcocystis neurona.

To make a passive immunity vaccine comprising polyclonal antibodies, the16 (±4) kDa antigen and/or 30 (±4) kDa antigen or one or more epitopestherefrom are injected into a suitable host for preparing theantibodies, preferably the host is a horse, swine, rabbit, sheep, orgoat. Methods for producing polyclonal antibody vaccines from thesehosts are well known in the art. By way of brief example, the antigen isadmixed with an adjuvant such as Freund's complete or the less toxicTiterMax available from CytRx Corp., Norcross, Ga., which thenadministered to the host by methods well known in the art.

The passive immunity vaccine can comprise one or more monoclonalantibodies against one or more epitopes of the 16 (±4) kDa antigenand/or 30 (±4) kDa antigen. Methods and hybridomas for producingmonoclonal antibodies are well known in the art. While monoclonalantibodies can be made using hybridoma technologies well known in theart, the monoclonal antibodies against the antigen can also be madeaccording to phage display methods such as that disclosed in U.S. Pat.No. 5,977,322 to Marks et al., which is hereby incorporated herein byreference. Equinized antibodies against the antigen can be madeaccording to methods which have been used for humanizing antibodies suchas those disclosed in U.S. Pat. Nos. 5,693,762 and 5,693,761 both toQueen et al., which are hereby incorporated herein by reference. A phagedisplay kit that is useful for making monoclonal antibodies is theRecombinant Phage Antibody System available from Amersham PharmaciaBiotech.

To make the vaccines of the present invention, the genes encoding the 16(±4) kDa antigen and/or 30 (±4) kDa antigen are identified usingmonoclonal antibodies against the 16 (±4) kDa antigen and/or 30 (±4) kDaantigen to screen a cDNA expression library made from mRNA isolated fromSarcocystis neurona. Since expression of certain Sarcocystis neuronaproteins is stage specific, not only are cDNA expression libraries madefrom mRNA isolated from Sarcocystis neurona grown in culture but cDNAlibraries are also made from mRNA isolated from Sarcocystis neurona atvarious stages of development, i.e., the merozoite, sporocyst, andsarcocyst stages. Methods for screening cDNA expression libraries withmonoclonal antibodies are described in Molecular Cloning: A LaboratoryManual, Second Edition, edited by Sambrook et al. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). The expressionlibrary can be a plasmid-based expression library that uses a pUC, pUR,pEX or a lambda-based expression library. Preferably, the library is aZAP EXPRESS vector (available from Stratagene, La Jolla, Calif.) whichis a hybrid lambda-plasmid vector used to construct cDNA libraries. RNAis isolated using a Stratagene RNA isolation kit and cDNA is made usingthe ZAP EXPRESS cDNA Synthesis kit (available from Stratagene). Thelibrary is screened using monoclonal antibodies against the 16 (±4) kDaantigen and/or 30 (±4) kDa antigen and the picoBLUE Immunoscreening kit(available from Strategene).

An important aspect of any vaccination program is to be able todistinguish animals vaccinated against a disease from animals infectedwith the disease. Therefore, the present invention further includesmethods that distinguish equids vaccinated with the vaccine of thepresent invention from equids infected with Sarcocystis neurona, orequids vaccinated with whole-organism Sarcocystis neurona vaccinepreparations, or equids never exposed to Sarcocystis neurona. In oneembodiment, to distinguish vaccinated equids from infected equids, abiological sample from an equid is tested for the presence of antibodiesagainst Sarcocystis neurona specific antigens that are in addition tothe 16 (±4) antigen and 30 (±4) kDa antigen which are induced by thevaccine. For example, Granstrom et al. in J. Vet. Diagn. Invest. 5:88-90(1993) identified by gel electrophoresis followed by Western blot eightSarcocystis neurona antigens; 70 kDa, 24 kDa, 23.5 kDa, 22.5 kDa, 13kDa, 11 kDa, 10.5 kDa, and 10 kDa, of which at least three (22.5 kDa, 13kDa, and 10.5 kDa) were common to all seven equids infected withSarcocystis neurona. Therefore, an equid that had antibodies against anyof the above Sarcocystis neurona antigens in addition to the 16 (±4) and30 (±4) kDa antigens would be infected with, or exposed to, Sarcocystisneurona whereas an equid that had antibodies against the 16 (±4) antigenand 30 (±4) kDa antigen but not against any one of the other Sarcocystisneurona antigens would be an equid that had been vaccinated with thevaccine of the present invention but was not infected with Sarcocystisneurona.

Therefore, in a Western blot embodiment consisting of Sarcocystisneurona antigens resolved by gel electrophoresis, a biological samplefrom a vaccinated equid would contain antibodies that bind only with the16 (±4) antigen and 30 (±4) kDa antigen whereas a sample from an equidinfected with, or exposed to, Sarcocystis neurona would containantibodies that bind with additional Sarcocystis neurona antigens. Theequine antibodies that are bound are identified by treating the blotwith labeled antibodies against equine antibodies. Preferably, the labelis selected from the group consisting of alkaline phosphatase,horseradish peroxidase, fluorescent compounds, luminescent compounds,colloidal gold, and magnetic particles. Methods for preparing andanalyzing Western blots are well known in the art. In a preferredembodiment, the Western blot is pretreated with non-equine antibodiesagainst a Sarcocystis sp. other than Sarcocystis neurona wherein thepretreatment prevents binding of equine antibodies to those antigenscommon to all Sarcocystis spp. which can be present in the sample. Thismethod is disclosed in Provisional Patent Application Ser. No.60/120,831, filed on Feb. 19, 1999, now U.S. Ser. No. 09/506,630, filedFeb. 18, 2000, which is hereby incorporated herein by reference.

In an enzyme-linked immunosorbent assay (ELISA) embodiment, a microtiterplate is provided containing a plurality of wells wherein a first wellor series of wells contains the 16 (±4) kDa antigen immobilized to thesurface therein, a second well or series of wells contains the 30 (±4)kDa antigen immobilized to the surface therein, and a third well orseries of wells contains another Sarcocystis neurona specific antigenimmobilized to the surface therein. Next, the biological sample is addedto the wells containing the bound antigens and antibodies againstSarcocystis neurona are allowed to bind to form an antibody-antigencomplex. The biological sample can be provided neat or in a limitingdilution series in a physiological solution. Unbound material in thesample is removed from the antibody-antigen complex by washing. Thecomplex is then reacted with a labeled antibody or labeled monoclonalantibody that binds to equine antibodies to form a secondantibody-antigen complex. The second complex can be detected when thelabeled monoclonal or polyclonal antibody is conjugated to a reporterligand such as horseradish-peroxidase or alkaline phosphatase.Alternatively, the second monoclonal or polyclonal antibody can beconjugated to reporter ligands such as a fluorescing ligand, biotin,colored latex, colloidal gold magnetic beads, radioisotopes or the like.Detection of the complex is by methods well known in the art fordetecting the particular reporter ligand. Therefore, a sample from anequid that had been vaccinated will produce antibodies against only the16 (±4) antigen and 30 (±4) kDa antigen whereas a sample from an equidthat is infected with, or exposed to, Sarcocystis neurona will containantibodies against the third antigen in addition to containingantibodies against the 16 (±4) antigen and 30 (±4) kDa antigen. ELISAwas developed by Engvall et al., Immunochem. 8:871 (1971) and furtherrefined by others such as Ljunggren et al. J. Immunol. Meth. 104:7-14(1987) and Kemeny et al., J. Immunol. Meth. 87:45-50 (1986). ELISA andits variations are well known in the art. The ELISA can be provided as akit for distinguishing vaccinated equid from unvaccinated equid, andfrom an equine infected with Sarcocystis neurona.

Since it is important to be able to test samples in the field in orderto distinguish equids infected with Sarcocystis neurona from equidsvaccinated with the vaccine of the present invention, the presentinvention further includes rapid immunodiffusion-based methods, theirdevices, and kits comprising the same. Therefore, the present inventioncan be provided with a kit that comprises any one of the methodsdescribed in U.S. Pat. No. 5,620,845 to Gould et al., U.S. Pat. No.5,559,041 to Kang et al., U.S. Pat. No. 5,656,448 to Kang et al., U.S.Pat. No. 5,728,587 to Kang et al., U.S. Pat. No. 5,695,928 to Stewart etal., U.S. Pat. No. 5,169,789 to Bernstein et al. U.S. Pat. No. 4,486,530to David et al., and U.S. Pat. No. 4,786,589 to Rounds et al. While theaforementioned disclose particular rapid immunodiffusion methods, thepresent invention is not to be construed to be limited to theaforementioned. It is within the scope of the present invention toembrace derivations and modifications of the aforementioned. Forexample, the 16 (±4) antigen and/or 30 (±4) kDa antigen are immobilizedto one area of a membrane and a third Sarcocystis neurona antigen isimmobilized to another area of the membrane in a device designed foranalyzing a biological sample. A biological sample is applied to themembrane which diffuses throughout the membrane. If the sample containsantibodies that form antibody-antigen complexes with all three antigens,the equid is infected with, or exposed to, Sarcocystis neurona. If thesample contains antibodies that form complexes with the 16 (±4) and/or30 (±4) kDa antigens and no antibodies that bind to the third antigen,the equid has been vaccinated with the vaccine of the present inventionbut is not infected with Sarcocystis neurona. Detection of theantibody-antigen complex is by a colorimetric method incorporated intothe device, by immersing the device into a solution that causes acolorimetric reaction, or by reacting with a labeled monoclonal orpolyclonal antibody conjugated to a reporter ligand.

Another method for distinguishing vaccinated equids from equids infectedwith, or exposed to Sarcocystis neurona is to provide as the vaccine theaforementioned fusion polypeptide wherein the polypeptide comprises amarker epitope that elicits an antibody in the vaccinated equid thatwould not normally be present in the equid. For example, the markerepitope could be from a pathogen that does not infect equids or asynthetic polypeptide that elicits an antibody in equids that would notnormally occur in equids. Therefore, if a sample from an equid containedantibodies against the marker epitope and the 16 (±4) antigen and/or 30(±4) kDa antigen, the equid was vaccinated with the vaccine of thepresent invention, whereas if the sample does not contain antibodiesagainst the 16 (±4) antigen and/or 30 (±4) kDa antigen, the equid isinfected with Sarcocystis neurona. The sample is tested according to anyof the aforementioned diagnostic methods.

In a method further still for distinguishing vaccinated equids frominfected equids, the vaccine of the present invention consists of apolypeptide that comprises a subset of the total epitopes on the 16 (±4)antigen and/or 30 (±4) kDa antigen. Therefore, in an equid vaccinatedwith the above polypeptide vaccine, antibodies are produced against onlythose epitopes on the polypeptide whereas in an equid infected withSarcocystis neurona, antibodies are produced against all of theepitopes. Thus, a sample from an infected equid will produce antibodiesthat binds the vaccine polypeptide and the full-sized antigen whereas asample from a vaccinated equid will produce antibodies that will bindthe vaccine polypeptide but not the full-sized antigen. Theantibody-antigen or antibody-polypeptide complex can be detected bymodifying any of the aforementioned diagnostic assays.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLE 1

This example is to demonstrate the preparation of monoclonal antibodiesthat recognize 16 (±4) kDa antigen and/or 30 (±4) kDa antigen ofSarcocystis neurona.

Sarcocystis neurona was cultured on equine dermal cell line cultures astaught in Example 3 or on bovine monocyte cell cultures as taught byGranstrom et al., J. Vet. Diagn. Invest. 5:88-90 (1993). Sarcocystisneurona merozoites were harvested and the 16 (±4) kDa antigen and/or 30(±4) kDa antigen were purified by methods known to the art for purifyingantigens, i.e., the 16 (±4) kDa antigen and/or 30 (±4) kDa antigen werepurified from merozoites by two-dimensional polyacrylamide gelelectrophoresis. Then the purified antigens are used to make monoclonalantibodies according to the methods in Antibodies, A Laboratory Manual,eds. Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988), well known to those skilled in the art as a sourcefor methods for making polyclonal and monoclonal antibodies.

BALB/c mice are immunized with an initial injection of 1.0 μg of the 16(±4) kDa antigen and/or 30 (±4) kDa antigen per mouse mixed 1:1 withFreund's complete adjuvant. After two weeks, a booster injection of 1.0μg of antigen is injected into each mouse intravenously withoutadjuvant. Three days after the booster injection the mouse serum ischecked for antibodies to the 16 ±4 kDa and/or 30 ±4 kDa antigens. Ifpositive, a fusion is performed with a mouse myeloma cell line. Mid logphase myeloma cells are harvested on the day of fusion, checked forviability, and separated from the culture medium by low-speedcentrifugation. Then the cells are resuspended in serum-free Dulbecco'sModified Eagle's medium (DMEM).

The spleens are removed from the immunized mice and washed three timeswith serum-free DMEM and placed in a sterile Petri dish containing 20 mlof DMEM containing 20% fetal bovine serum, 1 mM pyruvate, 100 unitspenicillin, and 100 units streptomycin. The cells are released byperfusion with a 23 gauge needle. Afterwards, the cells are pelleted bylow-speed centrifugation and the cell pellet is resuspended in 5 ml 0.17M ammonium chloride and placed on ice for several minutes. Then 5 ml of20% bovine fetal serum is added and the cells pelleted by low-speedcentrifugation. Afterwards, the cells are resuspended in 10 ml DMEM andmixed with myeloma cells to give a ratio of 3:1. The cell mixture ispelleted by low-speed centrifugation, the supernatant fraction removed,and the pellet allowed to stand for 5 minutes. Next, over a period of 1minute, 1 ml of 50% polyethylene glycol (PEG) in 0.01 M HEPES pH 8.1 at37° C. is added. After 1 minute incubation at 37° C., 1 ml of DMEM isadded for a period of another 1 minute, then a third addition of DMEM isadded for a further period of 1 minute. Finally, 10 ml of DMEM is addedover a period of 2 minutes. Afterwards, the cells are pelleted bylow-speed centrifugation and the pellet resuspended in DMEM containing20% fetal bovine serum, 0.016 mM thymidine, 0.1 hypoxanthine, 0.5 μMaminopterin, and 10% hybridoma cloning factor (HAT medium). The cellsare then plated into 96-well plates.

After 3, 5 and 7 days half the medium in the plates is removed andreplaced with fresh HAT medium. After 11 days, the hybridoma cellsupernatant is screened by an ELISA assay. In this assay, 96-well platesare coated with the appropriate 16 (±4) kDa antigen or 30 (±4) kDaantigen. One hundred μl of supernatant from each well is added to acorresponding well on a screening plate and incubated for 1 hour at roomtemperature. After incubation, each well is washed three times withwater and 100 μl of a horseradish peroxide conjugate of goat anti-mouseIgG (H+L), A, M (1:1, 500 dilution) is added to each well and incubatedfor 1 hour at room temperature. Afterwards, the wells are washed threetimes with water and the substrate OPD/hydrogen peroxide is added andthe reaction is allowed to proceed for about 15 minutes at roomtemperature. Then 100 μl of 1 M HCl is added to stop the reaction andthe absorbance of the wells is measured at 490 nm. Cultures that have anabsorbance greater than the control wells are removed to 2 cm² culturedishes, with the addition of normal mouse spleen cells in HAT medium.After a further three days, the cultures are rescreened as above andthose that are positive are cloned by limiting dilution. The cells ineach 2 cm² culture are counted and the cell concentration adjusted to1×10⁵ cells per ml. The cells are diluted in complete medium and normalmouse spleen cells are added. The cells are plated in 96-well plates foreach dilution. After 10 days, the cells are screened for growth. Thegrowth positive wells are screened for antibody production; thosetesting positive are expanded to 2 cm² cultures and provided with normalmouse spleen cells. This cloning procedure is repeated until stableantibody producing hybridomas are obtained. Then the identified stablehybridomas are progressively expanded to larger culture dishes toprovide stocks of the cells.

Production of ascites fluid is performed by injecting intraperitoneally0.5 ml of pristane into female mice to prime the mice for ascitesproduction. After 10 to 60 days, 4.5×10⁶ cells are injectedintraperitoneally into each mouse and ascites fluid is harvested between7 and 14 days later.

An alternate method for screening hybridomas for antibody production isas follows. Sarcocystis neurona is heat-denatured in 0.5 M Tris (pH 7.4)with 10% SDS, 20% glycerol and 5% 2-mercaptoethanol. The denaturedantigens are separated by SDS-polyacrylamide gel electrtophoresis in a12-20% (v/v) linear gradient gel with a 4% (v/v) stacking gel. Theseparated antigens are electrophoretically transferred to Western PVDFmembranes at 100 volts for 1.5 hours, then 150 volts for 0.5 hours. Themembranes are then blocked overnight in 1% by volume bovine serumalbumen in 0.5% Tween-Tris buffered saline (Blocking buffer). The blotsare air-dried and stored frozen. Prior to use, the membranes areincubated with bovine serum albumin and Sarcocystis cruzi antibodies inBlocking buffer at a range of 1:10 to 1:100 ratio for two hours.Afterwards, the membranes are washed in 0.5% Tween-Tris buffered salineand then incubated with monoclonal antibodies from the various hybridomaclones. The membranes are developed as disclosed in the prior art, e.g.,Granstrom et al., J. Vet. Diag. Invest. 5:88-90 (1993) or Antibodies, ALaboratory Manual, eds. Harlow and Lane, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1988).

Hybridomas that successfully produce monoclonal antibodies againstvarious epitopes of the 16 (±4) kDa antigen and 30 (±4) kDa antigen areexpanded as above, and used to make monoclonal antibodies for theantigen-based immunoassay and for identifying cDNA library clones inExample 2 that contain Sarcocystis neurona DNA which express either the16 (±4) and/or 30 (±4) kDa antigens.

In the foregoing procedure, monoclonal antibodies against particularepitopes of the identifying antigens are produced.

EXAMPLE 2

This example shows the preparation of a cDNA library that expresses the16 (±4) kDa antigen and/or 30 (±4) kDa antigen of Sarcocystis neurona.The methods for making and screening cDNA expression libraries are wellknown to those skilled in the art and are described in MolecularCloning: A Laboratory Manual, Second Edition, edited by Sambrook et al.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).The monoclonal antibodies made as in Example 1 are used to screen thelibrary for clones that express the 16 (±4) kDa antigen and/or 30 (±4)kDa antigen.

This example provides a simplified method for the isolation,excystation, and culture of Sarcocystis species using opossums as amodel. The method is an improvement over the isolation, excystation andculture methods of the prior art and is useful for producing antigensfrom various Sarcocystis neurona strains for subunit vaccines, formaking monoclonal and polyclonal antibody vaccines, and attenuated andkilled whole organism vaccines.

Opossums are humanely killed and their intestines screened forSarcocystis spp. oocysts. In addition, Sarcocystis oocysts collectedfrom wild grackle (Quiscalus sp.) fed possums and oocysts collected fromwild-caught cowbird (Molothrus ater) fed opossums in the inventors'laboratory can be used. A 2-cm segment of mid-small intestine from eachanimal is removed and washed with 0.01 M phosphate-buffered saline, pH7.4 (PBS). A scraping of mucosa is observed at 100× magnification usinga Nikon Optiphot-2 microscope to determine the presence or absence ofoocysts. Feces from the large intestine is removed from each positiveanimal and tested for the presence of Sarcocystis spp. sporocysts andother parasite ova by sucrose flotation according to Sloss et al., InVeterinary Clinical Parasitology, Iowa State University Press, Ames.Iowa, (1994), p. 198. The small intestine is flushed with PBS to removecontents and slit lengthwise. The mucosa is scraped off with a glassslide and ground in a Dounce homogenizer. The slurry is transferred to aconical tube and washed three times with PBS by centrifugation for 10minutes at 500× g. The pellet is resuspended in 3 volumes ofpepsin-NaCl-HCl (0.65% pepsin w/v, 0.86% NaCl w/v, 1% concentrated HClv/v) and incubated at 37° C. for 1.5 hours with frequent mixing. Theslurry is washed 3 times with PBS as above and the pellet stored inHank's balanced salt solution (HBSS) plus penicillin (100 units/ml),amikacin (100 μg/ml), and amphotericin B (1.25 μg/ml) until further use.A 1 to 3 ml aliquot of the semidigested mucosa is concentrated bycentrifugation for 10 minutes at 500× g. The pellet is suspended in 15ml of 2.6% sodium hypochlorite solution, stirred for 1.5 hours at roomtemperature, and washed once with PBS as above.

The improvement in the excystation and culture of Sarcocystis sp. overthe prior art is the mechanical excystation step as set forth below. Thewashed sodium hypochlorite pellet is suspended in 15 ml 10% trypsin inalkaline chelating solution (ACS) which is a solution that consisted of100 mM NaCl, 3 mM KCl, 9 mM Na₂HPO₄, 3 mM Na-citrate, 0.5 mM Na₂EDTA,0.1% glucose, 0.3% HEPES, 100 units penicillin, and 1.25 μg/mlamphotericin B, and incubated 1.5 hour at 37° C. After washing once withPBS as above, a drop of the pellet is compressed between sterile slidesand shearing forces are applied by moving the slides back and forth. Thematerial on the slides is washed with cell medium into flasks ofconfluent equine dermal cells (ATCC CCL-57, freely available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209) in Dulbecco's modified Eagle's medium (DMEM; availablefrom GIBCO a division of Life Technologies, Bethesda, Md.) plusL-glutamine, 6% heat-inactivated fetal bovine serum, penicillin (100units/ml), amikacin (100 μg/ml), and amphotericin B (1.25 μg/ml).Sarcocystis neurona isolated from neural tissue of EPM-affected horsescan be passaged continuously long term on this cell line. Before andafter inoculation, equine dermal cells are grown at 37° C. with 5% CO₂,with medium changed every other day for 7 days and weekly thereafter.After inoculation, cultures are observed weekly for evidence of cellulardamage due to Sarcocystis spp. replication and for the presence ofextracellular merozoites using an Olympus CK2 inverted microscope.Positive cultures are confirmed by Romanowsky (modifiedGiemsa-Wright)-stained cytospin of infected cells using a ShandonCytospin 3 centrifuge and a Wescor 7100 Aerospray slide stainer.Separate sterile pipettes are used to add or withdraw media from eachflask containing each separate strain to eliminate the possibility ofcross contamination.

The above improved method enabled obtaining viable organism from 7opossums that had Sarcocystis sporocysts detected in the feces. All ofthese opossums were adult males, 6 of which were from the same Michiganfarm on which two horses had been diagnosed with histopathologicallyconfirmed EPM. Each opossum harbored a million or more oocysts in thesmall intestinal mucosa; however, fewer than two sporocysts per gram offeces were observed in each when feces from the large intestine wastested by sucrose flotation. Ascarid, strongyle, tapeworm, Caillariasp., Physaloptera sp. eggs, or a combination of these eggs were alsoobserved in the wild-caught animals.

In the improved method, processing the mucosa with a Dounce homogenizerand subsequent pepsin-NaCl-HCl digestion broke down tissues but did notdisrupt Sarcocystis oocysts, many of which were still attached to tissuefragments (Murphy and Mansfield, 1999). Further digestion with sodiumhypochlorite freed most of the oocysts and released many sporocysts.Three chemical excystation methods as set forth in Example 4 wereattempted. All were effective in breaking down the oocyst walls andweakening the sporocyst walls, but none to few excysted sporocysts weredetected afterward. However, mechanical excystation as performedaccording to the improvement shown herein proved to be most effective,especially with the 10% trypsin ACS pretreated sporocysts.

Processed small intestine from the first opossum isolate refrigerated inHBSS plus penicillin, streptomycin, and amphotericin B remainedcontaminated with bacteria. Inoculation of dermal cells with thiscontaminated material resulted in cell death. Culture and sensitivitytesting proved the contaminating organism to be Alcalcigens sp. Amikacin(100 μg/ml) was substituted for the streptomycin in the mucosalpreparation and in all subsequent solutions, including the cell growthmedia. Amikacin killed the contaminant and no bacterial contamination ofany subsequent isolates using the penicillin-amikacin-amphotericinB-enhanced media.

Successful culture of merozoites from the first opossum isolate occurredin 13 of 15 flasks into which sporocysts pretreated with 10% trypsin inACS and mechanically excysted by the improved method herein wereinoculated. In contrast, 4 flasks each were inoculated with the threedifferent regimes of chemically excysted sporocysts without mechanicalexcystation as shown in Comparative Example 1. All remained negativeexcept for 1 trypsin-ACS- and 1 bile-trypsin-pretreated inoculum.

Thus, the trypsin-ACS/mechanically excysted sporocysts made as above,infected more efficiently than those prepared by chemical methods; eachflask became positive by visual examination at about 10 to 30 sitesbetween about 5 to 15 days after inoculation. In contrast, thetrypsin-ACS pretreated sporocysts became positive in culture 14 daysafter inoculation and at one site, and the bile-trypsin-pretreatedsporocysts became positive in culture 26 days after inoculation at onlyone site. Successful culture was further confirmed by Romanowsky-stainedcytospin of infected cells. All flasks negative for merozoites visuallyand by Romanowsky-stained cytospin of cells were discarded eight weeksafter inoculation because longer term culture did not result in morepositive flasks in preliminary trials. The mechanical excystation methodhas been used for all subsequent opossum isolates. The six additionalisolates became positive using microscope visualization from 6 to 14days after inoculation at many sites in each flask. All strains isolatedfrom these seven opossums have grown well long term (six months orlonger).

Sporocysts collected from six specific pathogen-free opossums fedwild-caught cowbird were successfully excysted and grown in equinedermal cell culture in our laboratory using this technique as weresporocysts thought to be Sarcocystis falcatula from opossums fedwild-caught grackle (these were wild-caught opossums testing negativefor Sarcocystis by fecal flotation for three weeks prior to infection).The cowbird isolates have grown well long term in equine dermal cells.Marsh et al., J. Parasitology 83:1189-1192 (1997) have shown that anequine-derived Sarcocystis neurona isolate grew highly efficiently longterm in equine dermal cells. The grackle-fed opossum isolate grew inequine dermal cells but only for a brief time, 3 to 8 weeks in threedifferent infection trials. Although the cell line was not effective forlong-term growth of this Sarcocystis sp., the excystation method andinitial culture were successful.

This example shows that multiple isolates of merozoites have beensuccessfully cultured from opossum-derived Sarcocystis spp. oocystsusing the improved method of digestion followed by manual excystation.Long-term growth of all opossum Sarcocystis spp. should be possibleusing the improvement and the appropriate cell line. Equine dermal cellswork well for Sarcocystis neurona, but other cell lines may be moreuseful for other Sarcocystis spp. A more complete understanding of thelife cycle of Sarcocystis neurona and, therefore, of the factors thatdetermine exposure of horses should be possible using the opossumisolates derived from the above improved excystation and culturemethods.

EXAMPLE 4

This example provides three chemical excystation methods for preparingSarcocystis sp. oocysts. The chemically prepared samples were comparedto samples prepared by the improved method shown in Example 3.

Samples were prepared as in Example 3 except that after washing thepellet that had been suspended in 2.6% sodium hypochlorite, the sampleswere treated with either (1) 10% trypsin in ACS, (2) 10% bile and 2%trypsin in HBSS (Speer et al., J. Protozoology 33:486-490 (1986)), or 5%sodium taurocholate and 2% trypsin in PBS (Speer et al., ibid.). All thesamples were incubated at 37° C. and 5% CO₂. The chemical methodsprovided poor results even though the methods were effective in breakingdown the oocyst walls and weakening the sporocyst walls.

Flasks inoculated with samples from the three above chemically excystedsporocysts remained negative except for one trypsin-ACS- and onebile-trypsin-pretreated inoculum. The trypsin-ACS-pretreated sporocystsbecame positive in culture 14 days after inoculation in one site and thebile-trypsin-pretreated sporocysts became positive in culture 26 daysafter inoculation at one site. In contrast, the improved method as wasshown in Example 3 was more efficient. Each flask became positive byvisual examination at many sites 5 to 15 days post-inoculation.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the Claims attached herein.

1. A method for producing a passive immunity vaccine against Sarcocystisneurona in horses, comprising: (a) providing Sarcocystis neurona 16 kDaantigen and Sarcocystis neurona 30 kDa antigen, as determined by SDSpolyacrylamide gel electrophoresis; (b) immunizing one or more mammalswith the Sarcocystis neurona 16 kDa antigen and the Sarcocystis neurona30 kDa antigen, in admixture with an adjuvant, to produce antibodiesagainst the Sarcocystis neurona 16 kDa 4antigen and antibodies againstthe Sarcocystis neurona 30 kDa antigen; (c) removing serum from the oneor more immunized mammals; (d) isolating from the serum from the one ormore immunized mammals the antibodies against the Sarcocystis neurona 16kDa antigen and the antibodies against the Sarcocystis neurona 30 kDaantigen; and (e) administering the antibodies to the 16 kDa antigen andantibodies to the 30 kDa antigen, isolated in step (d), together tohorses as the passive immunity vaccine against Sarcocystis neurona. 2.The method of claim 1, wherein the one or more mammals immunized in step(b) are selected from the group consisting of horses, swine, rabbits,sheep, and goats.
 3. The method of claim 1, wherein the adjuvant in step(b) comprises Freund's complete adjuvant.